1. Field of the Invention
The present invention relates generally to the field of molecular biology of herpes simplex viruses and vaccine technology. More specifically, the present invention relates to a means of improving gene therapy for diseases such as cancer by mutating herpes simplex virus type 1 thymidine kinases and uses thereof.
2. Description of the Related Art
The herpes simplex virus thymidine kinases (HSV-TKs).sup.1 are the pharmacological targets of most herpesvirus treatments (1,2), and more recently, HSV-1 TK has been utilized as a suicide gene therapeutic for cancer in combination with ganciclovir (3,4). The basis for these uses is their ability to specifically phosphorylate anti-herpesvirus nucleoside drugs such as acyclovir (ACV), ganciclovir (GCV) and 5-bromovinyldeoxyuridine (BVDU) (1,2,5). This targeting is based primarily on the differences in substrate specificity compared to the cellular TKs. The HSV-1 TK has a much broader range of substrates which include most pyrimidine nucleosides, many guanosine derivatives (e.g., ACV or GCV), and most purine and pyrimidine nucleoside triphosphates (6-9). HSV-TK also possesses a thymidylate kinase (TMPK) activity, but this activity is restricted to only deoxypyrimidine monophosphate substrates (7-9).
Proteolytic mapping studies of HSV-1 TK with the photoactive TMP analog, [.sup.32 P]5N.sub.3 dUMP, identified a region of the thymine base binding site inclusive in the peptide Ile.sup.112 -Tyr.sup.132 (10). This report, and others (7,8,11,12), concluded that the thymine base of TMP and thymidine bind in one shared site. This was subsequently confirmed in comparisons of two X-ray crystal structures of HSV-1 TK with bound thymidine or TMP (13, 14). Two initial X-ray crystal structures of HSV-1 TK have been published (13,14), one with bound thymidine or ganciclovir (13) and the other with thymidine, 5-iodo-deoxyuridine monophosphate or a complex with TMP and ADP (14). Subsequent structures have been reported with bound acyclovir, penciclovir and other nucleoside drug substrates and inhibitors (15,16). Within the pyrimidine base binding site, all structures have indicated that hydrogen bonding between Gln-125 of HSV-1 TK and the N3 and O4 atoms of the pyrimidine base was evident (13-15). In the complex with ganciclovir or acyclovir, Gln-125 was shown to form hydrogen bonds with the N1 and O6 atoms of the guanine base of GCV (13,15,16).
The prior art is deficient in lack of improved mutants of herpes simplex virus type 1 thymidine kinases useful in treating cancers in gene therapy techniques so as to maximize therapeutic efficacy and minimize untoward side effects. Increasing and/or modifying the desired substrate specificity for HSV-thymidine kinase would ameliorate these side effects. The present invention fulfills this long-standing need and desire in the art.